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It is obvious in the research, prepared all mediums of diets are harmed with trichogramma generation and put there eggs. But it is observed dying because of inconvenience medium of diets for developing parasite generation. According to diet mediums component development of trichogramma parasite was rather prolonged when G.melonnella hemolymphy and inorganic salt's quantity was more in diets medium. In fact quantity ofprotein and oil is more in the structure of hemolymphy as well as it is considered convenient for development ofparasite maggot.
References:
Thus, from above nominated artificial diet mediums, fifth diet medium (E) pupae hemolymph of G.melonnella (Ej) 45,5%, inor-ganicsalts (E2) 13,5%, chicken yolk (E3) 20,5%, milk (E4) 20,5% composed medium of diets is defined as a suitable one for normal nourishment and development of trichogramma generation in order to rear trichogramma parasite.
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Кимсанбаев. Х. Х., Жумаев Р. А. Renewing and rearing technology of Bracon hebetor Say in Biolaboratory; Материалы VIII-оймеждународной научно - практической конфе-ренции молодых исследователей, г. Волгоград, - 2014. - С. 257-259. Li Li-ying, Liu Wen-hui 1997. Rearing Trichogramma spp, with artificial diets, containing hemolymph of different insects. Parasitoids and predators (insecta) of agricultural and forestry arthropod pests - 335-337. (In China).
Li Li-ying, Liu Wenhui, Chen Chaoshian, Han Shityou, Shin Jiachi, Du Hansun, Feng shuyi, 1997. In vitro rearing of Trichogramma spp and Anastatus sp in artificial "eggs" and the methods of mass production. Parasitoids and predators (insecta) of agricultural and forestry arthropod pests - 344-357. (In China).
DOI: http://dx.doi.org/10.20534/ESR-16-9.10-13-16
Mamadaliyeva Nodira Isakovna, Post-doctoral student Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: [email protected] Saatov Talat Saatovich, Professor, Head of laboratory Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: [email protected] Umerov Oybek Ilyasovich Junior researcher Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: [email protected]
Study on effect of a-lipoic acid on phospholipid composition of rat cardiac tissue under hypobaric hypoxia
Abstract: Hypoxia is a rather frequent pathological or borderline state caused by the reduction of oxygen concentrations in the ambient environment or dysmetabolism of oxygen in an organism. The simulated chronic hypoxia similar to the one at the altitude of 7,000 m, caused changes in total sum of phospholipids in rat cardiac tissue and proportions of some phospholipids. Pharmacocorrection of the changes with berlithion containing a-lipoic acid was found to normalize levels of some rat cardiac tissue phospholipids.
Key words: cardiac tissue, hypobaric hypoxia, phospholipid.
Introduction [3, 49-50; 4, 10]. In many cases damage of integrity of membrane
Hypoxia is a rather frequent pathological or borderline state- bilayer's lipid phase accounts for fails indicating significant role oflip-caused by the reduction of oxygen concentrations in the ambient ids in pathogenesis of hypoxic conditions [5, 31-32; 6, 84]. environment or dysmetabolism of oxygen in an organism [1; 2183]. Changes in the lipid composition of cardiac tissue membranes
Hypoxia is capable of causing diseases independently and modi- under hypoxia are known to take place because of hypoenergetic fying courses of other diseases, primarily, cardio-vascular ones [2; condition, excessive lipid peroxidation [7, 140-141; 8, 413-415] 634-635]. and modification of membrane structures under the effect of cyto-
The findings from numerous recent studies aiming at elucidation toxic metabolites [9; 177]. Disorders in cardiac bioelectrical activity, of a mechanism underlying the onset and progression ofpathological onset and progression of arrhythmia and hypertension [10; 177], processes associated with hypoxia of cardio-vascular system are the atherosclerosis [11; 188], myocardial hypertrophy, ischemic heart evidence for the fact that changes in a cell membrane's structure and disease and myocardium infarctions [12; 380] are among the con-function are the significant link in the chain of the pathology's onset sequences taking place [13; 199-200].
The work was initiated to study effect of chronic hypobaric hypoxia on concentrations of several fractions of the rat cardiac tissue phospholipids and their total sum with subsequent pharmaco-correction of changes by means of a-lipoic acid as a constituent of berlithion 300 (Menarini Group, Berlin-Chemie AG, Germany).
Materials and methods
Hypobaric hypoxia was simulated in 108 senior outbred male rats weighing 250-280g by means of an altitude test chamber [14;3] with preset pressure of308 mm Hg similar to the one at the altitude of7,000m by 8-hour exposures for 10, 20 and 30 days. The animals exposed to hypoxia were divided into two groups; no correction of hypoxia was performed in the first group of rats, in the second group berlithion was intraperitoneally administered in the dose of 100 mg/kg of rat body mass (hypoxia+berlithion).
The control group kept in habitual conditions ofthe animal facility included 36 rats divided into three groups by 12 animals in a group administered with a physiological solution for 10, 20 and 30 days, respectively. All animals were slaughtered by decapitation with their hearts being weighted and homogenized in liquid nitrogen after the slaughter. Extraction of total lipids and their purification from nonlipid impurities was performed by Folch's method [15; 499] with recommendations of Kates [16; 74] by means of chloroform-methanol mixture (2:1). Extraction was performed within 60 minutes with
regular shaking of flasks at the room temperature. The method allows sufficiently complete extraction of tissue lipids. Crude lipid extract (CLE) was stored at 0-4 °C to measure amounts of phospholipids and their fractionation. Following mineralization of the samples, estimation of phospholipid absolute amounts and separate fractions was performed by amount of phosphorous in them with colorimetric assay ofthe non-organic phosphorus by Vaskovsky et al. [17; 129-141].
Fraction composition ofphospholipids was analyzed by one-dimensional ascending chromatography in thin silica gel on 13x18 cm glass plate in the system of solvents (chloroform-methanol-acetic acid-water, 16:4:1:4). The solvent from achieved, the plates were taken from the chamber and dried for fractions of phospholipids to be developed in iodine vapor.
Results and discussion
Prolonged hypoxia both in human beings and animals is known to induce adaptive changes on various levels of cell organization, which allow sustaining proper level of homeostasis in an organism under oxygen deprivation [18, 4-6; 19, 598].
Induced in rats, the simulated chronic hypoxia similar to the one at the altitude of 7,000 m, was found to cause increase in total sum ofphospholipids (Fig.1) and oppositely directed alterations in proportions of phospholipid fractions (Table 1-4).
Figure I.Total sum of phospholipids in rats under chronic regular hypoxia (similar to the one at the altitude of 7,000m)
Figure 1 demonstrates that total sum of phospholipids in hy-poxia exposed rats tended to increase, though the changes were not significant all the time (P<0.05). Thus, if on the 10th day of experiment amounts oftotal phospholipids in hypoxia exposed rats not subjected to correction were significantly different from those in the controls (39.109 mmol/kg versus 36.078 mmol/kg of raw tissue), on the 20th day of experiment the parameter was higher in all hypoxia exposed rats than in the controls. Total phospholipids in the cardiac tissue of control animals on the 20th day of experiment were 36.046 mmol of
phospholipids/kg of raw tissue; 42.704 and 38.805 mmol of phos-pholipids/kg of raw tissue in the first and second groups of hypoxia exposed rats, respectively. On the 30th day of experiment parameters in the first group remained unchanged (42.436 mmol of phospholipids/kg of raw tissue in hypoxia exposed rats versus 36.108 mmol of phospholipids/kg of raw tissue in the controls). Despite correction, the situation in the second group of rats heightened (40.647 mmol of phospholipids/kg of raw tissue in hypoxia exposed rats versus 36.108 mmol of phospholipids/kg of raw tissue in the controls).
Table 1. — Levels of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) in rat cardiac tissue under correction of hypoxia
Control Hypoxia Hypoxia + berlithion
10 exposures
lysophosphatidylcholine 0.46±0.02 1.41±0.06 1.02±0.05
phosphatidylcholine 14.57±0.67 13.47±0,61 14.34±0.6
20 exposures
lysophosphatidylcholine 0.46±0,02 2.5±0,1 1.46±0.07
phosphatidylcholine 14.57±0,67 14.7±0,65 13.6±0,64
30 exposures
lysophosphatidylcholine 0.46±0,02 2.48±0.08 1.66±0.05
phosphatidylcholine 14.57±0.67 14.82±0.73 14.87±0.65
Levels of lysophosphatidylcholine (LPC) in all hypoxia exposed rats were higher than those in the controls within all periods of study (Table 1). Following administration ofberlithion, levels of lysophosphatidylcholine clearly reduced, that is, a-lipoic acid can be concluded to suppress increase in LPC levels, thus demonstrating efficacy of this type correction for hypoxia.
Though insignificantly, levels of phosphatidylcholine (PC) in hypoxia exposed rats regardless of hypoxia duration was found lower than those in the controls (Table 1). Thus, on the 10th day of
experiment average PC levels in the first group of rats were 13.47 ± 0.61 mmol/kg of raw tissue, PC levels on the 20th and 30th days of experiment were 14.37 ± 0.65 and 14.82 ± 0.73 mmol/kg ofraw tissue, respectively. In the second group of hypoxia exposed rats on the 10th day of experiment average PC levels were 14.34 ± 0.60 mmol/kg of raw tissue, 13.96 ± 0.64 mmol/rg of raw tissue could be seen on the 20th and 30th days of experiment. The findings demonstrate efficacy of berlithion for phosphatidylcholine in hypoxia of moderate duration (10 exposures).
Table 2. - Levels of phosphatidylethanolamine (PE) and diphosphatidylglycerol in rat cardiac tissue under correction of hypoxia
Control Hypoxia Hypoxia+berlithion
10 exposures
phosphatidylethanolamine 10.75±0.51 13.6±0.061 11.8±0.54
diphosphatidylglycerol 4.93±0.24 2.9±0.13 2.79±0.13
20 exposures
phosphatidylethanolamine 10.75±0.51 15.15±0,68 13.4±0.61
diphosphatidylglycerol 4.93±0.24 3.19±0.14 3.15±0.14
30 exposures
phosphatidylethanolamine 10.75±0.51 14.46±0.59 13.69±0.58
diphosphatidylglycerol 4.93±0.24 2.95±0.14 2.79±0.13
The findings from study of phosphatidylethanolamine (PE), another significant fraction of neutral phospholipids, deserve special mention. Data in Table 2 demonstrate effect of berlithion on PE levels manifesting specifically on the 20th and 30th days of experiment. As to diphosphatidylglycerol, the levels of this lipid most regrettably remained unchanged in all hypoxia exposed rats regardless of terms. The findings are indicative of low sensitivity in phospholipids of mitochondrial inner membranes to antioxidants and Krebs cycle stimulation. In our study hypoxia was found to facilitate reduction in the levels of diphosphatidylglycerol in rat cardiac tissues, being indicative of its biosynthesis inhibiting and intensification of degradation; the phenomena impossible to be improved by phar-macocorrection with a-lipoic acid.
Our findings from studying levels of phosphatidylserine (PS)
and phosphatidylinositol (PI) are of special interest, since increase in the levels of these phospholipids could be observed within all periods and in all hypoxia exposed animals (Table 3). It may well be that, regardless of number of exposure, rapid increase of PS levels is both qualitatively and quantitatively necessary to sustain membrane asymmetry, as well as for adequate function of various ion pumps, such as, Ca2+ pumps and sodium-potassium pumps, in which phosphatidylserine is directly involved. Increase in PS levels may be facilitated both by increase in its biosynthesis and reduction in PS decarboxylation to phosphatidylethanolamine (PE) associated with the decline in PE methylation to phosphatidylcholine (PC) under the effect of N-metyltransferase to explain the increase in PE levels and the reduction in PC levels versus the values in the controls [20; 212-213].
Table 3. - Levels of phosphatidylserine (PS) and phosphatidylinositol (PI) in rat cardiac tissue under correction of hypoxia
Control Hypoxia Hypoxia+berlithion
10 exposures
phosphatidylserine 1.19± 0.06 1.43±0.06 1.52±0.07
phosphatidylinositol 1.88±0.09 2.51±0.11 1.99±0.09
20 exposures
phosphatidylserine 1.19± 0.06 1.55±0.07 1.64±0.07
phosphatidylinositol 1.88±0.09 2.93±0.13 2.17±0.1
30 exposures
phosphatidylserine 1.19± 0.06 1.42±0.06 1.79±0.08
phosphatidylinositol 1.88±0.09 2.45±0.08 2.34±0.1
In our study, under chronic hypoxia levels ofphosphatidylino-sitol (PI) were found to increase in hypoxia exposed rats not receiving berlithion and to decrease upon its administration. Following administration of berlithion, PI levels in short term exposure to hypoxia (10 exposures) were compared with those in the controls (1.99 ± 0.09 versus 1.88 ± 0.09 mmol/kg of raw tissue), after 20 and 30 exposures PI levels were significantly different. Berlithion was found to facilitate reduction in PI levels as compared with those in the rats undergoing no correction, but the values were not comparable with those in the controls. Increase in PI levels under in chronic hypoxia is thought to be an adaptive mechanism to optimize energy resources with ATP deficiency. Hypoxia causes oxidative phosphor-
ylation uncoupling to stimulate anaerobic glycolysis and providing PI synthesis by substrates, while stimulation of phosphatidylinositol synthase accelerates adjoining of inositol to cytidin diphosphate-diacylglycerol. After correction of chronic hypoxia with berlithion, stimulation of energy metabolism and optimization of conditions for oxidation of pyruvate and Acetyl-CoA took place resulting in reduction of activation of PI energy system and in PI decline. In addition, berlithion facilitates complete oxidation of carbohydrates in the myocardium, utilization of glucose-6-phosphate for oxidation and synthesis of ATP, but not of inositol with glucose-6-phosphate as its precursor. In addition, pharmacocorrection stabilizes intracellular machinery facilitated by phosphatidylinositol [21; 26].
Levels of sphingomyelin (SPH) in hypoxia exposed rats not undergoing pharmacocorrection were significantly different from those in the controls within all periods of observation. After 10 exposures to hypoxia, berlithion was found to facilitate reduction in SPH absolute content, while in under prolonged hypoxia irrespectively of pharmacocorrection with berlithion SPH levels tended to increase.
Conclusions
To sum up, the simulated chronic hypoxia similar to the one at the altitude of 7,000 m, caused increase in total sum of phospholipids in rat cardiac tissue; levels of some phospholipids, such as lysophosphatidylcholine, sphingomyelin, phosphatidylethanol-
The findings demonstrate efficacy of berlithion for sphingomyelin under hypoxia of moderate duration (10 exposures) (Table 4).
Following the pharmacocorrection with berlithion, regardless of hypoxia duration, levels of phosphatidic acid (PA) tended to decrease, yet remaining significantly higher than those in the control group within all periods of observation.
amine, phosphatidylserine, phosphatidylinositol and phosphatidic acid were found to increase, while levels of diphosphatidylglycerol markedly decreased. Pharmacocorrection of the changes with berlithion containing a-lipoic acid was found to normalize levels of some rat cardiac tissue phospholipids.
Table 4. - Levels of sphingomyelin (SPH) and phosphatidic acid (PA) in rat cardiac tissue under correction of hypoxia
Control Hypoxia Hypoxia+berlithion
10 exposures
sphingomyelin 1.86±0.09 2.26±0.1 2.03±0.09
phosphatidic acid 0.44± 0.02 1.51±0.07 0.91±0.04
20 exposures
sphingomyelin 1.86±0.09 2.06±0.09 2.1±0.1
phosphatidic acid 0.44± 0.02 1.3±0.06 0.92±0.04
30 exposures
sphingomyelin 1.86±0.09 2.25±0.09 2.15±0.09
phosphatidic acid 0.44± 0.02 1.59±0.06 1.36±0.06
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